We shall use the methods of protein crystallography and molecular biology to establish the structural basis for the functioning of HIV reverse transcriptase (RT) including its RNase H domain, integrase, tat and rev with the long term goal of finding novel pharmaceuticals for the treatment of HIV infections. A major focus of our RT studies will be the initiation of reverse transcription include the nature of abortive DNA synthesis and the translation to elongation synthesis. Our assay of the initiation of reverse transcription using tRNA/3/Lys and various length virion RNA templates will be developed further for high throughput screens for large chemical compound libraries to find inhibitors. Compounds that inhibit initiation but not elongation will be studied biochemically and structurally to ascertain which aspect of initiation is being affect. To investigate the role of p51, study the mechanism of RNase H and make complexes with tRNA, we shall study a fragment HIV RT heterodimer containing only the connection and RNase H domains of the p66 subunit and co-crystallize it with suitable ligands. We propose to engineer and make an active, monomeric p51 subunit and co-crystallize it with dideoxy terminated primer-template (DNA-DNA, RNA-RNA and DNA-RNA) and dNTP. Both in vivo and in vitro made tRNA/3/Lys associated with RNA templates of different lengths will be co-crystallized with full length RT as well as the fragment RT heterodimer. The crystal structures of an HIV RRE RNA fragment will be determined and of an HIV TAR dodecamer will be refined, both at 1.3 A resolution. We shall ascertain the influence of divalent metal ions on these RNA structures and on specific tat and rev peptide binding as well as establish the reason for the differences between the X- ray and NMR TAR structures. Further, tat and rev peptide complexes with these RNAs will be crystallized and their structures established. Crystallographic studies of both full length HIV integrase and the homologous transposase, TN552, complexed with suitable DNA substrates are aimed to produce mechanistic insights.